Membrane, process and system for isolating virus from solution

ABSTRACT

A composite membrane and process utilizing the membrane which is capable of selectively removing particles such as viral particles from a solution such as a protein solution is provided. The membrane comprises a porous membrane substrate, a surface skin having ultrafiltration separation properties and an intermediate porous zone between the substrate and the skin which intermediate zone has an average pore size smaller than that of the substrate. The intermediate zone is free of voids which break the skin and which directly fluid communicate with the substrate. The composite is capable of a log reduction value of at least 3 (99.9% removal) of particles selectively from solution.

BACKGROUND OF THE INVENTION

This invention relates to a membrane, process and system for removingparticles such as virus particles from solutions such as aqueous proteinsolutions effectively, selectively and reproducibly. More specifically,this invention relates to a composite asymmetric membrane having aspecific microstructure which is useful in a process or system forremoving virus at a log retention value of between about 3 and 8, i.e.,about 99.9 to 99.999999% removal of particles from solution.

Virus represent a potential contaminant in parenteral and othersolutions containing a protein which are derived from either wholeorganisms or mammalian cell culture sources. Currently several chemicaland physical methods exist to inactivate virus. These methods are notgeneric to all virus equally and some operate at the expense of proteinactivity. For example, heat pasteurization is used in solutions whereprotein denaturization can be minimized through the addition ofstabilizers. In the biotechnology industry, strategies have been adoptedthat combine several inactivation or removal steps in the downstreamprocess to maximize virus removal capability and protein recovery. Theoperations used are generally those operations optimized to purify theparenteral product and are validated for their virus removal capability.Thus, virus removal is a by-product of normal operation. Finally, at theend of the process, steps such as chromatography, filtration or heat maybe added to increase overall virus clearance. This strategy has twoshortcomings; (1) the virus clearance of these operations may not applyto putative virus that cannot be assayed; and (2) the virus clearance ofthe process needs to be monitored continually.

Ultrafiltration membranes have been proposed to separate virus fromprotein in solution. The ideal membrane would retain virus on the basisof its size and allow smaller proteins to pass. Indeed, ultrafiltrationmembranes are used in the biotechnology industry for this purpose.However, present asymmetric ultrafiltration membranes lack theresolution and reproducibility to perform an optimized virus-proteinseparation. Typically, asymmetric ultrafiltration membranes that areporous enough to pass economically useful percentages of protein, lackthe consistency and high level of virus retention to obtain optimumperformance that does not require continuous monitoring andrevalidation.

U.S. Pat. No. 4,808,315 describes a hollow fiber membrane with a uniquepore structure that is useful in the removal of virus from proteinsolutions. The membrane is not an asymmetric skinned ultrafilterpossessing a surface retention mechanism. Rather it retains virusparticles within its structure. It is described as a novel porous hollowfiber membrane which is characterized by such a unique porous structurethat the inner and outer membrane surfaces have an in-a-plane averagepore diameter of 0.01 to 10 microns and the porous membrane wall has anin-a-plane porosity of not less than 10% measured in every planeperpendicular to a radial direction of the annular cross-section of thehollow fiber membrane, wherein the in-a-plane porosity exhibits at leastone minimum value between the inner and outer membrane surfaces.

U.S. Pat. No. 4,824,568 discloses a process for forming an asymmetricskinned membrane on a porous support. The patent does not disclosewhether the membrane is useful for the selective removal of virus from aprotein-containing solution, nor does it disclose what modificationswould be required to obtain a microstructure useful for reproducibly andselectively removing virus particles from protein-containing solutions.

An asymmetric ultrafiltration membrane system that can recover more than95% of commercially significant proteins and can be validated having alog reduction value of at least about 3 logs of virus particles on thebasis of size (retention increasing monotonically as a function of virusparticle size) would offer a significant improvement over thoseavailable commercially today. This membrane and the system utilizing themembrane could then be used confidently to remove putative virus of anysize reproducibly and conveniently without the need for costlymonitoring and revalidation.

In addition, such a membrane could be utilized in other applicationswhere it is desired to remove small particles from solution such as inthe electronics industry.

SUMMARY OF THE INVENTION

The present invention is based upon the discovery that a particularasymmetric composite membrane structure having a skin possessingultrafiltration separation properties, a porous substrate and a porousintermediate zone is particularly useful for selectively isolating virusfrom a protein-containing solution. The thickness of the intermediatezone is larger than a thickness where the intermediate zone becomescollapsed or non-uniform and smaller than that where voids typical ofultrafiltration membranes are formed. The membrane is formed by castinga polymer solution containing between about 10 and 21% polymer onto amicroporous membrane. The cast polymer solution then is converted to aporous ultrafiltration skin and a porous intermediate zone by immersingthe coated membrane into a liquid which is miscible with the solventcomponent of the polymer solution but is a non-solvent for the polymercomponent of the polymer solution. Proper selection of the immersionliquid and temperature is important to obtain the combination of highvirus retention and high protein passage. The ultrafiltration skin andintermediate zone are characterized by small pores which provide amolecular weight cut off of between about 5×10² and 5×10⁶ Daltons. Bythe term "cut-off" as used herein is meant at least 90% removal ofspecies having a molecular weight at or higher than the stated cut-offmolecular weight. The intermediate zone is free of voids which form abreak in the skin and which cause fluid to communicate directly with theporous substrate. The coating concentration in the polymer solutioncoating and the coating thickness is controlled so that the thickness ofthe final dry intermediate zone is porous and is free of voids whichextend from the skin to the membrane substrate. It has been found thatthe composite membranes produced by this process having an intermediatezone which is free of voids normally found in ultrafiltration membranes,are uniquely capable of selectively isolating a virus by filtration froma protein-containing solution with selectivity and reproducibilityhigher than that obtained via conventional membrane casting techniques.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the coating step employed in the present invention.

FIGS. 2a, 2b, 2c, 2d and 2e are schematic diagrams of alternativeseparation systems of this invention.

FIG. 3 is a graph of the log reduction value of PhiX174 and the sievingcoefficient of human serum albumin as a function of the thickness of theintermediate porous zone.

FIG. 4 is a graph of the rejection coefficient of proteins of varioussize as a function of their Stokes radius for membrane A of thisinvention and commercially available ultrafiltration membranes.

FIG. 5 is a graph of the log reduction values of particles as a functionof the square of the particle diameter.

FIG. 6 shows the log reduction value of PhiX174 as a function ofvolumetric flux of the membrane produced in Example 3.

FIG. 7 shows the log reduction value of PhiX174 as a function of theratio of the recirculation flow rate to filtrate flow rate of themembrane produced in Example 3.

FIG. 8 shows the log reduction value of PhiX174 as a function of thechannel aspect ratio.

FIG. 9 is an exploded view of an ultrafiltration unit which is utilizedin Example III.

FIG. 10 is a top view of an ultrafiltration unit and the first spacer ofFIG. 7.

FIG. 11 is a cross-sectional view of a rectangular channel of theapparatus of FIGS. 7 and 8.

FIG. 12 is a cross-sectional view of an ultrafiltration hollow fiberwhich can be utilized in the present invention.

FIG. 13 is a photomicrograph of a cross-sectional view of a typicalmembrane produced by the process of U.S. Pat. No. 4,824,568.

FIG. 14 is a photomicrograph of a cross-sectional view of the membraneproduced by the process of this invention.

FIG. 15 is a photomicrograph of an alternative composite membrane ofthis invention.

DESCRIPTION OF SPECIFIC EMBODIMENTS

The composite membrane of this invention comprises an asymmetric skinnedmembrane which functions as an ultrafiltration membrane having a uniquemicrostructure. The membrane of this invention is made by a processsimilar to that disclosed in U.S. Pat. No. 4,824,568 which isincorporated herein by reference but with additional requirements. Mostimportantly, the step of coating the porous substrate with polymersolution is effected under carefully controlled conditions onto amicroporous substrate to form a skin and an intermediate zone betweenthe exposed skin and the substrate which is porous and is free of voidswhich extend from the skin t the substrate. Secondly, the compositionsof the immersion liquid that controls the removal of polymer solvent andcoagulation of the polymer is an organic bath designed to lengthen thepolymer coagulation time. In addition, the method of coating the polymersolution onto the porous substrate must be carefully controlled to applythe desired polymer solution thickness uniformly without damaging thesubstrate and without disrupting the coating so that it remainsuniformly thick. The proper combination of intermediate zone thicknessand immersion bath composition leads to the desired microstructure andthe performance combination of virus particle retention and proteinpassage in the case of protein solutions containing virus.

The substrate component of the composite membrane is formed of asynthetic material having a substantially continuous matrix structurecontaining pores or channels of a mean pore size between about 0.05 and10 micrometers. The substrate can be a microporous membrane, a nonwovensubstrate, a woven substrate, or a porous ceramic. A wide variety ofpolymeric materials can be utilized as the membrane, woven substrate ornonwoven substrate. Examples of these polymers include: polyolefins suchas low density polyethylene, high density polyethylene, andpolypropylene; vinyl polymers such as polyvinyl chloride andpolystyrene; acrylic polymers such as polymethylmethacrylate; oxidepolymers such as polyphenylene oxide; fluoropolymers, such aspolytetrafluoroethylene and polyvinylidene difluoride; and condensationpolymers such as polyethylene terephthalate, nylons, polycarbonates andpolysulfones.

The skin and intermediate zones of the composite membrane are made froma polymer solution as described herein. Exemplary polymer solutions canbe produced from all of the polymer suitable for forming the poroussubstrate as set forth above and including solutions of polyvinylidenedifluoride, cellulose esters such as cellulose acetate, polyimides suchas polyethermide, polysulfones, such as polyethersulfone andpolysulfone, polyacrylonitrile and the like.

In one embodiment, the pore surfaces of the porous substrate are treatedwith a liquid protecting agent to minimize or prevent the polymersolvent employed in subsequent coating steps from attacking thesesurfaces and from penetrating into the membrane. In the case ofmicroporous membranes formed from polyvinylidene difluoride (PVDF), suchas Durapore® membranes marketed by Millipore Corporation, Bedford,Mass., it has been found that treatment with glycerine is suitable. Themembrane can run as a web over a rotating coating roll having its lowerportion immersed in a solution of glycerine or can be totally immersedin a glycerine solution.

Liquid protecting agents other than glycerine can be employed includingglycols such as ethylene glycol, propylene glycol, triethylene glycol,or the like. Usually, it is preferable to select an agent which ismiscible with water because this facilitates removal of the agent in awater bath often used in substrate fabrication to extract from thesubstrate solvents and other materials employed in forming thesubstrate. Those skilled in the art will know, or be able to ascertainusing routine experimentation, additional liquid protecting agents. Theliquid agents can be dissolved in solutions, such as alcohol solutions.This facilitates application of the agent and the alcohol can be removedby subsequent drying.

In general, an amount of agent is employed which is sufficient toprovide the preformed substrate with significant protection againstattack from the polymer solvents employed in forming the compositemembrane having ultrafiltration separation properties and to providesignificant protection against substrate penetration by such solvents.The higher concentration of agent is determined by practicalconsiderations. For example, it has been observed that too muchglycerine can result in lower adhesion of the ultrafiltration membranesubsequently formed. Cost of the agent is another practicalconsideration. In treating Durapore® membranes with glycerine, it hasbeen determined that a preferred treating solution comprises from about15% to about 40%, by weight, of glycerine in isopropanol. Treatingagents that are not liquids can also be employed. For example, watersoluble waxes, such as polyethylene oxides, can be melted and applied tothe microporous membrane and removed, if desired, subsequently in theprocessing with a warm water bath.

The treated substrate is dried to remove any carrier for the protectingagent, e.g., isopropanol. Drying can be accomplished by conveying thetreated membrane over heated rolls, passing it through a heatedconvection oven, or by other techniques.

A composite membrane having ultrafiltration separation properties isthen formed upon the treated substrate structure. This is effected bycoating a polymer solution onto the treated substrate and quicklyimmersing the coated substrate into a liquid which is miscible with thesolvent but is a non-solvent for the polymer. A particularly preferredpolymer for the ultrafiltration membrane is PVDF, particularly when themiCroporous substrate is formed from PVDF. Although usually desirable,it is not necessary to form the ultrafiltration membrane from the samepolymer forming the substrate. However, in the preferred formation ofthe composite membrane, the polymer forming the ultrafiltration membraneis the same as the polymer forming the microporous substrate.

Polymer solutions containing between about 10 and 21%, preferablybetween about 19 and 21% of PVDF in a solvent are employed in order toobtain a ultrafiltration skin of appropriate cut-off. Lower PVDFconcentrations lead to more open structures with slightly higher proteinpassage and lower virus retention. The most selective and retentivestructure is achieved using the 19 to 21% preferred PVDF concentration.

In the case of PVDF, the coating process is specifically designed touniformly deposit a layer of this polymer solution such that the finaldry thickness of the coating is between about 5 and 20 microns,preferably between about 5 and 10 microns. Typical knife over rollcoating methods, such as generally used to coat ultrafiltration (UF)casting solutions onto substrates, are not optimum for such thincoatings requiring precise thickness control. The knife edge must be setclose to the moving substrate to obtain coatings within this narrowrange. Such fine settings and adjustments are difficult to obtain due tofrictional resistance and the normal tolerance of the knife design. Thethickness variability of the substrate being transported under the knifeis on the order of the gap (i.e., the space between the fixed knifeposition and the substrate) that is to be maintained. This variabilitychanges the actual gap and thereby the coating thickness. Also, in thecase of a microporous membrane substrate thickness variability can causebreakage when the microporous membrane substrate catches the knife orthe frictional resistance becomes too great. The problem is worse foredge curling or scalloping; "floppy edges". When breakage does occur,the knife must be removed, cleaned and reset before continuing. Sincebreakage of the relatively weak -- compared to normal nonwovensubstrates -- microporous membrane substrate is common, efficiency isreduced.

In order to provide controlled reproducible ultrafiltration skin, a newcoating method is provided in accordance with this invention. As shownin FIG. 1, coating thickness is controlled by forming a nip between arotating drum 76 and a non-rotating rubber coated cylinder 72. Themicroporous substrate 74 is positioned on a support web 78 whichcontacts the backed drum or roll 76 which can be rotating. Interposedbetween the rubber coated cylinder 72 and the polymer solution 84 is aplastic film 82 secured to so as to wrap cylinder 72. This film 82 canbe polyethylene terephthalate or any other film that is not adverselyaffected by the polymer casting solvent and is strong enough towithstand the shear forces imposed on it. The plastic film 82 can extendseveral inches past the nip point 80 in the direction of web transportand functions as a smoothing film. That is, the film 82 function tosmooth the exposed surface of the cast polymer solution 83 which exposedsurface forms the skin in the final composite membrane of thisinvention. It has been found that the use of the cylinder 72 and film 82permits accurate control of the thickness of the cast film 83 whichresults in the elimination of undesirable voids in the intermediate zoneof the composite membrane.

In operation, casting solution 84 is fed to a reservoir on the web entryside of the nip point 80 of the rubber covered cylinder 72 and the drum76. The moving microporous substrate 74 drags solution under the nip 80analogous to journal bearing lubrication. A simplified analysis showsthat the coating thickness is proportional to the square root of webspeed, casting solution viscosity and length under the nip 80, i.e. the"footprint" of the rubber covered roll; and inversely proportional tothe square root of the pressure under the nip. The footprint iscontrolled by rubber hardness and the pressure forcing the cylinder 72against the drum.

In practice, solution viscosity and casting speed are set by membraneproperty requirements. The hardness of rubber coating 85 is chosenempirically to give the desired range of coating thickness. Pressure onthe cylinder 72 is then used to set and control the exact thicknessobserved Pressure is set by pneumatic cylinders 86 acting on the metalcore 87 of cylinder 72. By controlling the pressure to the pneumaticcylinders 86, the force on the core 87 is controlled. Coating thicknesscan then be varied by adjusting the inlet pressure to the pneumaticcylinders 86.

After the polymer solution has been precisely coated onto themicroporous substrate, the ultrafiltration membrane structure is formedby immersing the coated microporous substrate into a liquid which ismiscible with the polymer solvent but is a non-solvent for the dissolvedpolymer. A solution comprising 25 wt % glycerine dissolved in water isthe preferred liquid for composite membranes made from PVDF at thepreferred 19-21% solids concentrate, for example. Although other liquidssuch as monohydric alcohols, water, or mixtures thereof, can be used,optimal membrane properties are obtained when an organic containingwater bath is employed and preferably 25 wt % glycerine in water.

When the precipitation process occurs slowly such as greater than 0.5minutes, preferably about 0.65 to 1 minute, as is achieved with 25 wt %glycerine in water in a thin coating, a unique asymmetric morphology isobtained in the composite membrane. The composite membrane comprises askin with ultrafiltration separation properties, a microporous substrateand, in the case of PVDF, an intermediate zone between the skin and thesubstrate having a thickness between about 5 and 20 microns. Themorphology of the intermediate zone is characterized as a continuousmatrix structure usually associated with asymmetric microporousmembranes but of mean pore size that is substantially smaller and intothe ultrafiltration range.

Unlike conventional ultrafiltration membranes as well as those describedin U.S. Pat. No. 4,824,568, the structure of the coating on themicroporous substrate of this invention is characterized by the absenceof elongated voids extending through the intermediate zone from theexposed surface of the skin to the microporous substrate below theintermediate zone. This attribute permits the membrane described hereinto be useful in the retention of virus particles while maintaining theprotein passage properties characteristic of conventionalultrafiltration membranes. Structures containing infrequent small voidsalso can result from the process described herein especially at thelower solids content. However, these structures are satisfactoryprovided that the small voids are infrequent and appear below while notextending to the exposed surface of the skin. However, the preferredstructure is one that is a continuous matrix with voids absent. Thistype of structure is found at the preferred conditions described hereinand is shown FIG. 14. This structure is in contrast to the structure ofthe membrane disclosed in U.S. Pat. No. 4,824,568 as shown in FIG. 13.As shown in FIG. 15, the intermediate zone can contain infrequent largervoids. However these larger voids do not extend from the skin to thesubstrate as is true with the ultrafiltration membrane shown in FIG. 13.

After the membrane structure has formed, the composite web is prewashedby conveying the coated and precipitated web through a water bath.Contact time of approximately one minute in 25° C. water is sufficient.Drying can be performed by leaving the prewashed web to dry as singlesheets at room temperature. Alternatively, the web can be continuouslydried by conveying the web over a perforated roll. The interior of theroll is kept at subatmospheric pressure and a heated air stream (e.g.,140° F.) is impinged on the surface of the web. A typical speed for theweb over such a roll is 4 to 6 feet per minute.

It has been found that with PVDF polymers and 25 wt % glycerine-waterimmersion bath that an ultrafiltration membrane less than 5 micronsthick is less satisfactory because inadequate surface coverage of themicroporous substrate results and the virus retentive propertiesdeteriorate. In some cases a thin coating of less than 5 microns alsoforms a non-porous collapsed film rather than an open porous structure.When the thickness of the intermediate zone is greater than about 20microns, undesirable voids can appear in the intermediate zone which canpromote virus particle passage. With casting polymer solutions, theminimum and maximum acceptable thickness for the intermediate zone willvary slightly from the 5 to 20 micron range for PVDF. In any event, theintermediate zone is uniformly porous and is free of large voids whichextend from the skin to the substrate, unlike the voids found inconventional ultrafiltration membrane.

When the composite membrane of this invention has a skin surface whichis hydrophobic, it must be rendered hydrophilic in order to be useful intreating aqueous solutions such as aqueous protein solutions toselectively remove virus particles therefrom. A preferred process forrendering the membrane hydrophilic and low protein binding is disclosedin U.S. Pat. No. 4,618,533 which is incorporated herein by reference.Hydrophilization can be conducted by the process of U.S. Pat. No.4,618,533 as a continuous multistep process which transforms thehydrophobic membrane into a hydrophilic (water wettable) membrane. Inthat process, a roll of phobic membrane can be unwound and fed throughthe following sequence of process steps:

1. Alcohol wetting -- The membrane web is submerged or otherwisesaturated with an alcohol, typically isopropanol, to completely wet outand fill the porous structure.

2. Water exchange: The membrane web is submerged in a bath of water toreplace the alcohol.

3. Saturation with reaction solution: The water wet web is submerged inan aqueous bath of monomer and other reactants made up to the desiredcomposition. Exchange occurs in this bath and the web emerges filledwith an aqueous solution containing the reactants. As taught by U.S.Pat. No. 4,618,533, a composition comprising hydroxypropyl acrylate, acrosslinking agent and a suitable initiator, can be used.

4. Polymerization: the web is conveyed to and through a reaction chamberwhere polymerization of the reactants saturating the web takes place insitu.

Oxygen is excluded during the polymerization reaction. This can be doneby saturating the reaction chamber with an inert gas, nitrogen forexample; or by sandwiching the web between transparent sheets, such aspolypropylene.

5. Washing: After reaction, the web is transported through suitablewater wash steps such as submersion, spraying, etc.

6. Drying: The membrane is dried prior to winding and package asdescribed above. The preferred drying temperature is 300° F.

The hydrophilization process described in U.S. Pat. No. 4,618,533 ismodified herein to be used with the composite membrane of thisinvention. Any excess hydrophilization solution is removed from the skinsurface of the composite membrane so that the composite pore surface isnot covered with a layer of hydrophilic coating that bridges the pores.This can be achieved with, a stationary flexible rubber wiper, a niproll or the like to remove the excess surface liquid from the surfacesof the composite membrane.

The membranes of this invention are uniquely characterized by a logretention value (LRV; the negative logarithm of the sieving coefficient)for virus particles and other, particles that increases systematicallyand monotonically with the diameter of the particle; in the size rangeof interest for virus of 10 to 100 nm diameter. Empirically, the LRVincreases continuously with the size of the particle projected area (thesquare of the particle diameter). The absolute LRV can be adjusted by acorresponding adjustment in the membrane protein sieving propertiescreated by manipulating the coating solution solids content, orimmersion bath composition and temperature. The composite membranes ofthis invention having an intermediate zone of higher porosity have alower molecular weight cut off than the membranes of this inventionhaving an intermediate zone which is less porous. Where one is concernedwith removing small sized virus particles from protein solution,satisfactory LRV of at least about 3 is obtained with membranes having alower porosity intermediate zone. However, the molecular weight cut offis reduced thereby reducing protein recovery. Therefore, the user willchoose a composite membrane which gives satisfactory LRV and proteinrecovery. In any event, the membranes of this invention are capable ofproducing an LRV for virus of 3 and can extend to as high as about 8 orgreater where the virus particle size is between 10 and 100 nm diameter.In addition, the composite membranes of this invention are characterizedby a protein molecular weight cut off of between about 5×10² and 5×10⁷Daltons. In all cases, the empirical relationship with particleprojected area is retained. Log reduction values for virus particles(single solutes in solution; in absence of protein) depends upon thevirus particle size. Based upon the relationships illustrated in theexamples below, with small sized virus such as hepatitis an LRV ofgreater than about 3 can be obtained and with larger sized virus such asthe AIDS virus, a LRV of greater than 6 can be obtained.

Protein sieving properties can be adjusted to achieve performancetypical of conventional ultrafiltration membranes. These properties canbe adjusted through proper manipulation of the casting solution solidscontent and immersion bath composition and temperature as is customaryin the formation of ultrafiltration membranes. Higher temperaturespromote the formation of larger pores. Higher solids content promote theformation of smaller pores. The membranes of this invention can beformed with molecular weight cut-off values (the molecular weight of thesolute that is 90% rejected by the membrane under low polarizationconditions) of between 5×10² Daltons to 5×10⁶ Daltons.

The composite membrane of this invention can be in the form of a flatsheet or hollow fiber. In the case of a flat sheet, one surface of thesubstrate, is coated with the skin and intermediate zone. In the case ofhollow fiber, the inner or outer surface is coated with the skin andintermediate zone.

In one aspect of this invention, a process is provided for selectivelyseparating viral particles in an apparatus provided with channels or aplurality of hollow fibers wherein the feed stream is flowedtangentially across the skin. A similar device for separating bloodplasma into a high molecular weight plasma fraction and a low molecularweight fraction is disclosed in U.S. Pat. No. 4,789,482 which isincorporated herein by reference. In accordance with this invention, adevice is provided having a plurality of channels or hollow fibers andwhich is operated with a controlled flow rate ratio for a recirculationstream to a filter stream.

Referring to FIG. 2a, the protein solution contained in vessel 16 whichmay or may not contain a virus is introduced through conduit 10 by meansof a pump 12, passed through conduit 14 and directed to a filtrationstep 20 wherein protein solution is separated from virus by means of themembrane of this invention 22. The virus rich fraction which alsoincludes protein is recycled to vessel 16 by means of conduit 24. Theprotein rich fraction free of virus is recovered through conduit 26 bymeans of pump 28 and is directed to storage or to a point of use throughconduit 30.

Other process configurations are possible including the incorporation ofa diafiltration stream. Referring to FIG. 2b, diafiltration can be addedto the process depicted in FIG. 2a by introducing to vessel 16 a streamof buffer stored in reservoir 6 through conduit 2 by means of pump 4operated at a volumetric flowrate that is identical to that of pump 28.

In a second process configuration, a second membrane stage comprised ofa module 40 containing the membrane 42 of this invention, can beoperated in series with that described above to achieve higher overallvirus removal. Referring to FIG. 2c, the protein rich fraction of stream30 created from the process depicted in FIG. 2a is added to this secondstage. A recirculation stream 36 is introduced to the membrane module 40containing the membrane 42 of this invention by means of pump 34 andconduit 32. A virus enriched stream is recycled to pump 34 via conduit36. The protein rich stream from the first stage is introduced viaconduit 30 to the recirculation loop created by conduits 32 and 36 andpump 34. Protein rich fraction from the second stage free of virus isrecovered through conduit 38 by means of pump 44 and is directed tostorage or point of use through conduit 46. The volumetric flowratethrough pump 44 is equal to that through pump 28 and pump 12. If desiredpumps 28 and 44 can be replaced with throttle valves adjusted to achieveflowrates identical to that of pump 12 and each other.

In another embodiment, a multiple stage cascade an be used which isdescribed herein with reference to FIG. 2d. The protein solutioncontained in vessel 16 which may or may not contain a virus isintroduced through conduit 10 by means of pump 12 and passed throughconduit 14 and directed to a filtration step 20 wherein protein solutionis separated from virus by means of the membrane 22 of this invention.The virus rich fraction which also includes protein is recycled to pump12 by means of conduit 24. The protein rich fraction free of virus isrecovered through conduit 26 by means of pump 28 and is directed to thesecond filtration stage 40. A virus rich bleed stream 31 is providedfrom the recirculation loop comprised of conduits 14 and 24 and pump 12and is withdrawn via conduit 31 and pump 33. A second recirculationstream 36 is introduced to the membrane module 40 containing themembrane of this invention 42, by means of pump 34 and conduit 32. Virusenriched solution from module 40 is recycled to pump 34 via conduit 36.The protein rich stream from filtration module 20 is introduced viaconduit 30 to the recirculation loop created by conduits 32 and 36 andpump 34. Protein rich fraction from filtration module 40 free of virusis recovered through conduit 38 by means of pump 44 and is directed tostorage or point of use through conduit 46. Optionally, buffer can beintroduced to conduit 36 via conduit 50 to maintain constant volume inthe cascade. Additionally, to improve protein recovery, part of thefluid contained in the second recirculation loop can be recycled to thefirst recirculation loop and conduit 14 through conduit 52 by pump 54.In this configuration, the volumetric flowrates in streams 31, 50 and 52are identical and that of streams 10, 26 and 46 are also identical. Theamount of protein recovered and virus removed can be optimized bycontrolling the ratio of the flowrate of stream 31 to that of stream 46.It is to be understood that a plurality of filtration steps 20 can beutilized in series with appropriate feed and product conduits as shownwhereby virus rich stream 24 or 31 can be contacted in additionalfiltration steps 20 to produce additional protein rich fractions free ofvirus. These filtration steps can be operated with or without recyclestreams. In another embodiment, a dead ended process configuration canbe used which is shown herein with reference to FIG. 2e, wherein feed isintroduced into filtration step 20 and filtrate is removed throughconduit 26.

Referring to FIGS. 9 and 10, a typical structure utilizing rectangularhollow channels (FIG. 11) is shown and which can be utilized asseperation module in this invention. This general structure is disclosedin U.S. Pat. No. 4,540,492 which is incorporated herein by reference.

A filter unit 32 comprises, a first membrane 34, a second membrane 36, afirst spacer 38, and a second spacer 40 which, when joined together forma plurality of rectangular channels 48. The apparatus utilized for virusseparation can include a plurality of filter units 32 which arepositioned contiguous to each other and form a stack of filter units 32.Both the first membrane 34 and the second membrane 36 are of identicalconstruction and are formed from the composite membrane of thisinvention described above. Each membrane 34 and 36 is provided with twolongitudinal channels 42 and 44 and a widthwise channel 46. Thewidthwise channel 46 is not in fluid communication with either of thechannels 42 or 44. The first spacer 38 comprises of plurality ofchannels 48 which extend from edge 50 to edge 52 and outlet channel 54.When membranes 34 and 36 are contiguous to spacer 38, the edges 50 and52 coincide with the edges 56 and 58 respectively of membrane 36. Thesecond spacer 40 is provided with a protein solution inlet channel 60and virus-rich stream outlets 62 and 64. The second spacer 40 also isprovided with interior channel 68 which provide fluid communication withchannels 66, which in turn is in fluid communication with virus-richstream outlet 64. When spacer 40 is juxtaposed to membrane 36, edges 63and 65 coincide respectively with edges 56 and 58 of spacer 36. Thespacer strips 69 between channels 48 and spacer strips 71 between thechannels 66 are bonded to the next adjacent membrane (not shown) andprovide the necessary support for the membranes adjacent the channels sothat membrane flexibility is controlled to maintain the desired channelheight.

While the module structure shown in FIGS. 9 and 10 is useful in thepresent invention, it is to be understood that any module utilizing themembranes of this invention can be employed in the present invention solong as the operating conditions are controlled aS set forth below.

Referring to FIG. 10, the channels 48, of first spacer 38 are shown tooverlap into channels 42 and 44 of membrane 36. This overlap permitsintroducing a virus-containing protein solution into channel 42, passageof this solution lengthwise along channels 48 while being in contactwith membrane 36 and removal of virus-rich solution from channels 48through widthwise channel 44.

The modules described above, both thin channel and hollow fibers, can beoperated in a tangential flow mode at low volumetric conversions. Anoptimum module aspect ratio and corresponding optimum operatingconditions exist for the separation of virus particles from proteinsolutions. The optimum aspect ratio and operating conditions are inaccordance with those described in U.S. Pat. No. 4,789,482 which isincorporated herein by reference. The aspect ratio, L/h, to achieve highsolute recovery is defined by Equation 1:

    L/h=[K/12 ρμ-h/L.sub.p ]1/2                         Equation 1

K is a function of the ratio of the transchannel pressure drop to theaverage pressure in the channel. K is obtained experimentally by theprocedure set forth below. h is the channel height or the hollow fiberradius, ρ is the ratio of the recirculation stream flow rate, Q_(R), tothe permeate stream flow rate, Q_(p), μ is the viscosity of the incomingprotein stream being separated, L is the length of the channel or fiberand L_(p) is the membrane hydraulic permeability after the membrane iswet with the liquid to be ultrafiltered.

For channels of rectangular geometry having at least one wall formed ofa porous membrane, h is the distance between the membranes 90 and 92which define the height of the channel shown in FIG. 11. Generally h forthe rectangular channels is between about 0.0110 and 0.030 cm. In thepresent invention, the module aspect ratio, L/h, can range between about50 and 5000, preferably 200 to 300.

The module aspect ratio and module operating shear rate aresimultaneously optimized to achieve the desired selectivity at thelargest possible flux. Unlike conventional systems, this system does notoperate either at an excessively large shear rate or an excessively lowvolumetric flux, but at conditions which maximize the desiredselectivity which in the case of virus removal is the virus to proteinselectivity. An exact relationship has been found between the moduleaspect ratio and module operating shear rate which gives optimalseparation performance.

The maximum shear rate to be utilized in the apparatus is defined byequation 2: ##EQU1## wherein δ is the maximum shear rate to obtainoptimal selectivity performance.

The proportionality constant, D^(*), is obtained empirically by thefollowing procedure:

A prototype module is provided containing a plurality of thin channelsor hollow fibers of the type of ultrafiltration membrane to be utilizedin the final apparatus. The channels or fibers in the prototype can beof any dimension. A prototype having channels with an L/h of about 200has been found to be useful. Pumps and conduits are provided to form aflux stream to recover the low molecular weight component and to controlthe flow rate of the permeate, Q_(p). This permeate stream is recombinedin a feed reservoir with a recirculation stream comprising the highmolecular weight component obtained from the ultrafiltration channels orfibers. The recirculation stream flow rate (Q_(R)) is controlled. Aplurality of runs are made with the apparatus with either Q_(p) beingvaried and Q_(R) being maintained constant or Q_(R) being varied andQ_(p) being maintained constant. After each run, the separationperformed (selectivity) between the species of interest is measured.After each run the system also is thoroughly flushed such as with salineor water to remove all treated liquid from the system. A standardhydraulic permeability, e.g. water or saline, L_(p), is then measured bystandard methods. The value then is multiplied by the ratio of μ of thetreated liquid to μ of the standard fluid wherein μ is viscosity incentipoises and wherein L_(p) is used in equation 1. From theselectivity values obtained, the optimum selectivity is identified andthe Q_(p) and Q_(R) values which correspond to the optimum selectivitycan be determined. The selected optimum value refers to the Q_(p) andQ_(R) values when both the flux and selectivity are maximizedsimultaneously. The constant K can be calculated using the optimum valueof Q_(R) /Q_(p) using Equation 1.

Using the shear rate corresponding to the optimum Q_(R), the constantD^(*) then can be calculated from equation 2. D^(*) is a property of thesolution being ultrafiltered and for proteins is between about 1×10⁻⁷cm² /sec and 25×10⁻⁷ cm² /sec. The limits on ρ reflect the limits on theratio, Q_(R) /Q_(p). The upper limit is set by the size of therecirculation pump whereas the lower limit is set by the virus retentionwhich decreases at low values of Q_(R) /Q_(p), i.e. at high conversions,(the retained species become more concentrated as permeate is removed).For small viruses, the value of Q_(R) /Q_(p) greater than 20 to 1 arepreferred.

When an ultrafiltration device is designed and operated in accordancewith equations 1 and 2, the total membrane area in the device whichprovides optimal separation efficiency is give by equation 3:

    A=0.25 Q.sub.p L(1+K).sup.2 /D.sup.* K (μL.sub.p /h).sup.1/2 Equation 3

wherein A is the total membrane surface area.

Furthermore, the maximum transchannel pressure drop which can bemeasured directly is also given by equation 4 for optimal separationconditions:

    P.sub.C =2.0D.sup.* h.sup.1/2 /(1+K).sup.2 L.sub.p.sup.3/2 Lμ.sup.1/2 Equation 4

wherein ΔPC is the transchannel pressure drop.

Control of the concentration polarization in a tangential flow moduledepends both upon a combined match between the module aspect ratio andoperating shear rate Therefore, only a restricted range of moduledesigns and of operating shear rate are feasible. This results in anupper and lower limit for the factors, L/h, and ρ. The ratio ρ, ofrecirculation stream flow rate, Q_(R), to permeate stream flow rateQ_(p), is between about 5 and 100, preferably between about 10 and 50and most preferably 30.

Finally, with the optimal design, L/h, and operating conditions, δ, theratio of the transmembrane pressure drop at the channel outlet to thetransmembrane pressure drop at the channel inlet,

    b=TMP.sub.Outlet /TMP.sub.Inlet

is significantly different from 1.0. The value of b derived from thisinvention lies between 0.0 and 0.85, most typically 0.75.

As is shown in the examples, optimal virus separation and proteinrecovery is achieved under the conditions shown in U.S. Pat. No.4,789,482, however, the preferred values of aspect ratio and operatingconditions differ from those identified in the claims of that patent. Asshown in the examples, in the presence of protein, virus removal (LRV),is a function of both aspect ratio and the ratio of recirculationflowrate, Q_(R), to permeate flowrate, Q_(p). The range appropriate tovirus retention are shown to be an aspect ratio of between 100 to 1000and a value for ρ of between 20 and 200. These ranges are within thosedescribed in U.S. Pat. No. 4,789,482. However, in the absence ofprotein, the preferred aspect ratio is about 300 and the preferred valueof ρ is between 20 and 100. Aspect ratios of below 100 and above 1000both result in less virus retention; values of ρ below 20, i.e.conversions above 0.05, can be used, but result in a similar dramaticloss in virus retention.

In the presence of protein, such as human serum albumin, the virusretention is enhanced by protein polarization on the membrane surface.In this case, virus retention is much less affected by aspect ratio andis nearly independent of same as long as the value of ρ is above 10,i.e. as long as the conversion is below 0.1. Therefore, in the presenceof protein, the aspect ratio of between 100 to 500 is preferred and avalue of ρ of between 10 and 50 is most preferred.

Combining these two cases for general use, the values of aspect ratioand ρ are those described in U.S. Pat. No. 4,789,482, with the preferredaspect ratio of about 300 and the preferred value for ρ of between about20 and 30.

The following examples illustrate the invention and are not intended tolimit the same.

EXAMPLE I

A Durapore® microporous membrane having an average pore size of 0.22micrometers and marketed by Millipore Corporation, Bedford, Mass. wasemployed as the preformed microporous membrane. The membrane was treatedwith a 30% glycerine in isopropanol solution and dried.

A polymer solution containing 20.5% polyvinylidene difluoride (PVDF,Kynar 741, Pennwalt Corporation, Philadelphia, Pa.) and, 4.9% lithiumchloride in N-methyl Pyrrolidone (NMP) was cast onto the glycerninizedDurapore® microporous membrane at a speed of 15 feet per minuteutilizing the apparatus illustrated in FIG. 1, the coated membrane wasthen immersed in a 25 wt % glycerine in water bath maintained at atemperature of 7° C. The length of the polyester smoothing film of thecoating process described with reference to FIG. 1 is approx. 2-3inches. The air exposure between the coating polyester film andimmersion bath was 2 inches. After casting, the composite membrane wasimmersed in a water bath maintained at 25° C. for one minute and wassubsequently dried by conveying the prewashed web over a perforateddrying roll having subatmosphere pressure and a heated air stream (140°F.) impinging on the surface of the web which was moving at 6 feet perminute.

The general procedure used to render the membrane hydrophilic is thatdescribed in U.S. Pat. No. 4,618,533 which is described above. For themembrane used in this example, the reactant aqueous solution contained4% hydropypropyl acylate (HPA), crosslinking agent and free radicalinitiator. The hydrophobic membrane was sequentially and continuouslyconveyed through alcohol, water and reactant both at 25 feet per minute.The excess reaction solution was removed by means of flexible rubberwiper blades. PolymerizatiOn of the crosslinked copolymer was initiatedby UV light with a wavelength of 254 nanometers applied to both sides ofthe web. The reactant saturated web had a residence time ofapproximately 5-10 seconds in the UV light. The hydropholized web waswashed in water to remove excess reactants and dried on a perforateddrum, the interior of which was held at subatmospheric pressure, whileair heated to 300° F. was imPinged on the surface.

During the coating operation, the rubber roll nip pressure as applied bythe pneumatic cylinders and the speed at which the microporous substratewas pulled through the nip were varied in these membranes in order toproduce intermediate porous zone thicknesses as measured by SEM from 5to 20 micrometers. The nip pressure was varied from 85 to 175 psi andthe speed from 6.5 to 15 feet per minute.

The membrane B produced was challenged separately and independently withtwo different solutions, one a solution containing only Phi X 174bacteriaphage in phosphate buffered saline (PBS) and a second solutioncontaining 0.25% human serum albumin in PBS spiked with Phi X 174bacteria phage.

As shown in FIG. 3, at a thickness of the dried hydrophobic intermediatezone of 5 micrometers, the intermediate porous zone is significantlycollapsed resulting in low solute permeability, Phi X 174 LRV is veryhigh, about 5 logs, and albumin sieving is very low at 48%. As thethickness of the intermediate zone is increased to 8 micrometers, thezone and the surface skin become more permeable resulting in asignificant increase in protein passage and a loss in Phi X LRV. As theintermediate zone thickness is increased further to 20 micrometers, thePhi X LRV decreases at a much lower rate and albumin passage isunchanged.

EXAMPLE II

This example illustrates that the composite membranes of this inventionare capable of retaining virus particles in the absence of protein atlog reduction values that are significantly better than membranes of theprior art possessing equivalent protein sieving characteristics.Additionally, the particle log reduction valves of membranes of thisinvention increase monotonically as a function of the particle diameter,a property not observed with membranes of the prior art.

A first membrane of this invention identified as Membrane A was preparedas follows:

A Durapore® microporous membrane having an average Pore size of 0.22micrometers and marketed by Millipore Corporation, Bedford, Mass., wasemployed as the preformed microporous membrane. The membrane was treatedwith a 30% glycerine in isopropanol solution and dried.

A polymer solution containing 19.8% polyvinylidene difluoride (PVDF,Kynar 741, Pennwalt Corporation, Philadelphia, PA) and, 5% lithiumchloride in methyl pyrrolidone was cast onto the glycerninized Durapore®microporous membrane at a speed of 15 feet per minute utilizing theapparatus illustrated in FIG. 1. the coated membrane was then immersedin a 25 wt % glycerine in water bath maintained at a temperature of 7°C. The length of the polyester smoothing film of the coating processdescribed with reference to FIG. 1 is approx. 2-3 inches and thepressure of the pneumatic cylinders is 150 psi. The air exposure betweenthe coating polyester film and immersion bath was 2 inches. Aftercasting, the composite membrane was immersed in a water bath maintainedat 25° C. for 1 minutes and was subsequently dried by conveying theprewashed web over a perforated drying roll having subatmospherepressure and a heated air stream (140° F.) impinging on the surface ofthe web which was moving at 6 feet per minute.

Membrane A was hydrophilized as described in Example 1.

The dried hydrophobic composite membrane had an intermediate porous zonethickness of 7.2 to 9.6 microns as determined by a scanning electronmicroscope (SEM).

A second membrane of this invention identified as Membrane C wasprepared as follows:

A Durapore® microporous membrane having an average pore size of 0.22micrometers and marketed by Millipore Corporation, Bedford, Mass. wasemployed as the preformed microporous membrane. The membrane was treatedwith a 30% glycerine in isopropanol solution and dried.

A polymer solution containing 19.8% polyvinylidene difluoride (PVDF,Kynar 741, Pennwalt Corporation, Philadelphia, Pa.) and, 5% lithiumchloride in methyl pyrrolidone was cast onto the glycerninized Durapore®microporous membrane at a speed of 15 feet per minute utilizing theapparatus illustrated in FIG. 1. The coated membrane was then immersedin a 25 wt % glycerine in water bath maintained at a temperature of 7°C. The length of the polyester smoothing film of the coating processdescribed with reference to FIG. 1 is about 2 inches and the pressuresupplied to the pneumatic cylinders is 150 psi. The air exposure betweenthe coating polyester film and immersion bath was 2 inches. Aftercasting, the composite membrane was immersed in a water bath maintainedat 25° C. for 1 minutes and was subsequently dried by conveying theprewashed web over a perforated drying roll having subatmospherepressure and a heated air stream (140° F.) impinging on the surface ofthe web which was moving at 6 feet per minute.

The composite membrane was rendered hydrophilic by the followingprocedure:

membrane C was hydrophilized similarly to Membrane A. The impingementdrying air temperature was 275° F. The aqueous reactant concentrationcontained 5.1% hydroxypropyl acrylate, crosslinking agent and freeradical initiator.

The dried hydrophobic composite membrane had an intermediate porous zonethickness of 8.5 microns as determined by a scanning electron microscope(SEM).

A third membrane of this invention identified as Membrane D was preparedas follows:

A Durapore® microporous membrane having an average pore size of 0.22micrometers and marketed by Millipore Corporation, Bedford, Mass. wasemployed as the preformed microporous membrane. The membrane was treatedwith a 30% glycerine in isopropanol solution and dried.

A polymer solution containing 19.9% polyvinylidene difluoride (PVDF,Kynar 741, Pennwalt Corporation, Philadelphia, Pa.) and, 4.9% lithiumchloride in methyl pyrrolidone was cast onto the glycerninized Durapore®microporous membrane at a speed of 15 feet per minute utilizing theapparatus illustrated in FIG. 1. coated membrane was then immersed in a25 wt % glycerine in water bath maintained at a temperature of 8° C. Thelength of the polyester smoothing film of the coating process describedwith reference to FIG. 1 is about 2 inches and the pressure supplied tothe pneumatic cylinder is 150 psi. The air exposure between the coatingpolyester film and immersion bath was 2 inches. After casting, thecomposite membrane was immersed in a water bath maintained at 25° C. for1 minute and was subsequently dried by conveying the prewashed web overa perforated drying roll having subatmosphere pressure and a heated airstream (140° F.) impinging on the surface of the web which was moving at4 to 6 feet per minute.

Membrane D was hydrophilized continuously with membrane A.

The dried hydrophobic membrane had an intermediate porous zone thicknessof 8.1-9.3 microns as determined by a scanning electron microscope(SEM).

Membrane A was compared with commercially available ultrafiltrationmembranes, PTHK, membrane PLMK membrane, both available from MilliporeCorporation of Bedford, and YM-100 membrane available from AmiconCorporation, Danvers, Mass. to determine the protein sievingcharacteristics as a function of protein size and operating flux at aconstant recirculation flow rate to achieve a shear of 1100 ¹ /_(sec).

At both 0.6 liters/meters ² /hr (LMH) and 6.0 LMH the protein sievingcharacteristics of the virus Membrane A is essentially equivalent tothat of a typical 100,000 dalton cut-off commercially availableultrafiltration membrane. In both cases, the virus Membrane A issubstantially tighter than the Millipore PLMK membrane of 500,000 daltoncut-off as shown in FIG. 4.

The log reduction values of the three membranes of this example setforth above were compared with commercially available YM-100 membraneavailable from Amicon Corp. of Danvers, Mass.; PTHK membrane availablefrom Millipore Corporation of Bedford, Mass. both shown previously tohave nearly identical protein sieving properties. Also included are twomembranes identified as PZHK#1 and PZHK#2 made in accordance withExample 2 of U.S. Pat. No. 4,824,568 and hydropholized as describedabove and the commercially available UltiPor 0.04 micrometer membraneavailable from Pall Corporation of East Hills, N.Y.

The log reduction value was determined by the following procedure. Eachmembrane was challanged with a solution containing the challangeparticle in phosphate buffered saline in a tangential flow cell underconditions of 1100 sec ⁻¹ shear and a flux of 3 liters per square meterper hour. Samples of filtrate and challange solution were analyzed forparticle concentration and the LRV calculated as the logarithm of theratio of the challange concentration to the filtrate concentration. Twochallange particles are bacterial phage, Phi X 174 and Phi 6 and areassayed by a plaque assay using their respective host bacteria. Adilution series was generated to determine concentration. The particlesare latex particles available from Seragen Diagnostics, Inc.,Indianapolis, Ind. These latex particles were stabilized with 0.1%Triton X-100 surfactant to avoid agglomeration. The latex particles wereassayed by first collecting via dead-ended vacuum filtration, 10-50 mlsof filtrate onto a 25 millimeter disc of 0.03 micron or 0.05 micronNucleopore filter available from Nucleopore Corp., Pleasanton, Calif. Arepresentative portion of the Nucleopore filter disc is mounted on anSEM stage and photomicrographs of in excess of 20 fields are recorded.The particles observed in these photomicrographs are counted todetermine the concentration of latex in each sample.

A comparison of the log reduction values of these membranes is shown inFIG. 5 and Table 1.

As shown in FIG. 5 and Table 1, only the membranes of this inventionwere capable of removing viral-sized particles from solution with a logretention value that increases monotonically as a function of particlediameter up to a value of 8.1 LRV for a 93 nm diameter particle.Commerically available ultrafiltration membranes of similar proteinsieving properties show LRV values that are nearly independent ofparticle diameter increasing only 1/2-1 log over the size rangemeasured. The membranes of this invention provide at least 3 to 4 ordersof magnitude improvement in particle removal for particles above 70 nmdiameter as compared to these commercially available ultrafiltrationmembranes. Additionally, as is shown in Table 1, the performance of themembranes of this invention is very reproducible.

When compared with PZHK#1 and PZHK#2, the improvements in the castingtechnology described herein over that of U.S. Pat. No. 4,824,568 haveled to 3-5 log performance improvement over the entire particle sizerange measured.

Finally, the PTHK and Ultipore membranes demonstrated a loss inretention of Phi X 174 in the presence of HSA protein as is shown inFIG. 5, suggestive of the fact that Phi X 174 adsorption is contributingsignificantly to the particle removal measured with these two membranes.In the presence of HSA, the LRV of Phi X 174 is increased from 3.0 logsto 3.7 logs due to protein concentration polarization with the MembraneA and the Membrane C virus membranes of this invention. Therefore, themeasured removal of particles is being accomplished primarily on thebasis of size.

                  TABLE I                                                         ______________________________________                                        PARTICLE LOG REDUCTION VALVES                                                            Phi X    67 nm     Phi 6  93 nm                                    MEMBRANE   (28 nm)  latex     (78 nm)                                                                              latex                                    ______________________________________                                        A          2.9      6.5       >7.5   8.2                                      C          3.0      6.7              8.0                                      D          3.1                >7.5                                            PTHK       2.2      <3.06     3.5    <3.5                                     YM-100     3.1      <3.4      3.3    3.9                                      Pall .04 micron                                                                          0.7      <3.3      4.2    4.2                                      PZHK #1    0.08     --        1.92   --                                       PZHK #2    0.025    --        0.14   --                                       ______________________________________                                    

EXAMPLE III

The membrane of this invention identified as Membrane A and prepared asdescribed above was tested to determine the effect of tangential flowoperation conditions upon the capability to retain Phi X 174 bacterialvirus.

The composite membrane was incorporated into an apparatus similar tothat shown in FIGS. 9 and 10 which had one module 32 and having channels2.4 inches long and 0.0078 to 0.0063 inch high.

A 0.25 wt. % Human Serum Albumin protein solution (Alpha Therapeutic)was prepared which included Phi X 174 bacterial phage having a 28 nmdiameter at pH 7.4. The solution was passed through the separationapparatus in order to determine LRV as a function of flux through themembrane and as a function of the ratio of the recirculation flow rateto filtrate flow rate. The results are shown in FIGS. 6 and 7.

As shown in FIG. 6, the retention of the phage increases slightly as afunction of transmembrane flux but the performance is reversible whenthe flux is returned to a low value, performance that is consistant withprotein concentration polorization.

The PVDF composite membrane of this invention was tested to determinethe effect of the ratio of recirculation flow rate to filtrate flow rateon LRV. As shown in FIG. 7, Phi X 174 retention is increased above thata dead end filtration device as the value of this flowrate ratio isincreased. Therefore, as either the recirculation flow rate is increasedor the conversion (the reciprocal of this flowrate ratio) is decreased,the Phi X 174 retention is improved over that measured in dead endedfiltration in which the recirculation flow rate is zero and theconversion is 100%. As can be seen in FIG. 7, the phage LRV isindependent of the ratio of recirculation flow rate to filtrate flowrate above a value of 25:1.

EXAMPLE IV

The membrane of this example, Membrane E, was tested in the apparatussimilar to those shown in FIGS. 9 and 10 which had one module 32 andhaving channels 2.4 to 11.0 inches long and 0.004 to 0.030 inches highsuch that the effect of channel aspect ratio on virus log reductioncould be determined.

Membrane E was prepared as follows:

A Durapore® microporous membrane having an average pore size of 0.22micrometers and marketed by Millipore Corporation, Bedford, Mass. wasemployed as the preformed microporous membrane. The membrane was treatedwith a 30% glycerine in isopropanol solution and dried.

A polymer solution containing 20% polyvinylidene difluoride (PVDF, Kynar741, Pennwalt Corporation, Philadelphia, Pa.) and, 5% lithium chloridein methyl pyrrolidone was cast onto the glycerinized Durapore®microporous membrane at a speed of 15 feet per minute utilizing theapparatus illustrated in FIG. 1. coated membrane was then immersed in a25 wt % glycerine in water both maintained at a temperature of 7° C. Thelength of the polyester smoothing film of the coating process describedwith reference to FIG. 1 is approx. 2-3 inches and the pressure suppliedto the pneumatic cylinder is 150 psi. The air exposure between thecoating polyester film and immersion bath was 2 inches. After casting,the composite membrane was immersed in a water bath maintained at 25° C.for 1 minute and was subsequently dried by conveying the prewashed webover a perforated drying roll having subatmosphere pressure and a heatedair stream (140° F.) impinging on the surface of the web which wasmoving at 4 to 6 feet per minute.

Membrane E was hydrophilized as described in Example 1.

The dried hydrophobic membrane had an intermediate porous zone thicknessof 6-10 microns as determined by a scanning electron microscope (SEM).

The results when challanged with a PBS solution containing Phi X 174both in the presence and absence of HSA are shown in FIG. 8. All testswere conducted at a channel shear rate of 1100 sec ⁻¹ and at a 3 literper square meter per hour flux. The channel aspect ratio has littleeffect on the retention of virus above a value of about 100.

EXAMPLE V

The membrane of this invention, identified as Membrane A and prepared asdescribed above, was used in the two stage system of FIG. 2d todemonstrate the performance in a system as could be employed inpractice. The two stage system was operated under tangential flowconditions at a recirculation shear of 1100 sec ⁻¹ and a volumetric fluxof 6 liters per square meter per hour in both stages. The volume offluid processed was 200 mls and the processing time in each case wasabout 5 hours.

The feed solution consisted of a 0.25% HSA in phosphate buffered salinewas spiked with a phage, in one case Phi X 174 and in the second casePhi 6 each at about 5×10⁷ pfu/_(ml). Samples were drawn in each streamand the results reported in Tables II and III, the stream numbersreferred to in Tables II and III are shown in FIG. 2d. The ratio of themeasured HSA concentration to that of the starting feed and the virusLRV value in each stream after 5 hours is reported. The HSAconcentration ratio is compared with values calculated based upon therejection coefficients presented in FIG. 4. Concentrations in eachstream matches closely the theoretical value indicating HSA recovery inaccordance with membrane properties. In the case of Phi X 174, 4.2 logand 4.8 log removal is measured in the effluent of stage 1 and 2respectively and a total of 5.6 log overall removal measured in thefinal processed fluid. In the case of Phi 6, no Phi 6 was measured inthe effluent of either stage. In both experiments, virus is recovered instream 31 withdrawn from the first stage recirculation streams.

                  TABLE II                                                        ______________________________________                                                 Theoretical   Measured  X174                                         Stream   HSA Conc      HSA Conc  LRV                                          ______________________________________                                        26       .92           .89       4.2                                          38       .90           .90       4.8                                          46                               5.6                                          31       1.8           2.0       0.6                                          52       2.0           1.4       0.7                                          ______________________________________                                    

                  TABLE III                                                       ______________________________________                                                 Theoretical   Measured  Phi 6                                        Stream   HSA Conc      HSA Conc  LRV                                          ______________________________________                                        26       .99           .89       >6.5                                         38       .90           .64       >6.5                                         46                               >6.5                                         31       1.8           .7         0.1                                         52       2.0           1.53      >6.5                                         ______________________________________                                    

We claim:
 1. A composite asymmetric membrane designed for selectivelyseparating particles having a size within a size range characteristic ofthe size of virus particles from a solution containing said particleswhich comprises a substrate having pores of an average size betweenabout 0.05 and 10 microns, a surface skin and an intermediate porouszone being positioned between said substrate and said skin, saidintermediate zone being porous and free of voids which extend from saidskin to said membrane substrate, said composite membrane having aprotein molecular weight cut-off of between about 5×10² and 5×10⁶Daltons and having properties for producing a log reduction value of atleast about 3 and said log reduction value being a monotonicallyincreasing function of particle diameter in the particle size rangebetween about 10 and 100 nanometer diameter.
 2. The composite membraneof claim 1 wherein said intermediate porous zone has a thickness betweenabout 5 and 20 microns.
 3. The composite membrane of claim 1 wherein thesubstrate is a microporous membrane.
 4. The composite membrane of claim3 wherein the intermediate porous zone has a thickness between about 5and 20 microns.
 5. The composite membrane of claim 4 wherein themembrane substrate is formed from polyvinylidene difluoride.
 6. Thecomposite membrane of any one of claims 1, 2, 3 or 4 in the form of aflat sheet.
 7. The composite membrane of any one of claims 1, 2, 3 or 4in the form of a hollow fiber and wherein said skin comprises an outsidesurface of said fiber.
 8. The composite membrane of any one of claims 1,2, 3 or 4 having pores with a hydrophilic surface.
 9. The compositemembrane of claim 3 wherein the membrane substrate is formed frompolyvinylidene difluoride.
 10. The composite membrane of claim 3 whereinsaid skin, said intermediate porous zone and said membrane are formedfrom polyvinylidene difluoride.
 11. The composite membrane of any one ofclaims 9, 5 or 10 having an intermediate zone thickness between about 5and 10 microns.
 12. A process comprising: selectively removing particlesof a size between about 10 and 100 nanometers from a solution at a logreduction value of at least about 3 by, in a first filtration step,passing said solution in direct contact with the skin of the compositemembrane designed for selectively separating particles having a sizewithin a size range characteristic of the size of virus particles from asolution containing said particles which comprises a substrate havingpores of an average size between about 0.05 and 10 microns, a surfaceskin and an intermediate porous zone being positioned between saidsubstrate and said skin, said intermediate zone being porous and free ofvoids which extend from said skin to said membrane substrate, saidcomposite membrane having a protein molecular weight cut-off of betweenabout 5×10² and 5×10⁶ Daltons and having properties for producing a logreduction value of at least about 3 and said log reduction value being amonotonically increasing function of particle diameter in the particlesize range between about 10 and 100 manometer diameter retaining saidparticles by said skin sufficient to form a particle-rich solution whileallowing solute in said solution substantially free of said particles topass through said composite membrane sufficient for causing the logreduction value of particle removal is a monotonically increasingfunction of the diameter of said particles.
 13. The process of claim 12wherein the particles are virus particles and said solution is a proteinsolution.
 14. The process of claim 12 wherein the intermediate porouszone of said membrane has a thickness between about 5 and 20 microns.15. The process of any one of claims 12, 13 or 14 wherein said compositemembrane is in the form of a flat sheet.
 16. The process of any one ofclaims 12, 13 or 14 wherein at least a portion of said particle-richsolution is recycled to directly contact said skin of said compositemembrane.
 17. The process of any one of claims 12, 13 or 14 wherein saidcomposite membrane is in the form of a flat sheet and at least a portionof said particle-rich solution is recycled to directly contact said skinof said composite membrane.
 18. The process of any one of claims 12, 13or 14 wherein said particle-rich stream is passed, in a secondfiltration step directly into contact with a skin of a second compositemembrane of claim 1 to produce a second particle-rich solution and asecond solution substantially free of said particles.
 19. The process ofany one of claims 12, 13 or 14 wherein said particle-rich stream ispassed in a second filtration step directly in contact with a skin of asecond composite membrane of claim 1 to produce a second particle-richsolution and a second protein solution substantially free of particlesand recycling at least one of said first or second particle-richsolutions to one of said filtration steps.
 20. The process of claim 12wherein the skin, intermediate porous zone and said membrane substrateare formed from polyvinylidene difloride.